PROJECT SUMMARY Signal transduction pathways important during development are frequently associated with disease. Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death. The Hedgehog (Hh) pathway is important for development and a Ras-Dyrk1b pathway downregulates autocrine Hh signaling in PDA. Wdr68 and Dyrk1b can physically interact and are important for multiple events in the zebrafish, including craniofacial development. Wdr68 and Dyrk1b are also important for edn1 expression for craniofacial development. Intriguingly, Wdr68 physically bridges signaling from MEKK1 to Dyrk1b suggesting the existence of Ras- MAP3K-Wdr68-Dyrk1b signal relay systems. However, while it is clear that Dyrk1b plays a key role in modulating Hh signaling in PDA, the role of Dyrk1b in Hh signaling during normal development is unknown. Likewise, the role of Wdr68 in Hh signaling is unknown. Furthermore, the genetic requirements and timing for wdr68 in edn1-mediated craniofacial development are still unclear. Therefore, the overall aim of this application is to better define the in vivo roles for Wdr68 and Dyrk1b. Our central hypothesis is that Wdr68 is part of an evolutionarily conserved transcriptional co-regulator complex that modulates multiple signaling events important during embryonic and adult life. The central hypothesis will be tested through three Specific Aims. First, we will test wdr68 and dyrk1b gene activity for roles in Hh signaling. We hypothesize that wdr68 and dyrk1b modulate Hh signaling during craniofacial development. The experimental approach will use in situ hybridization (ISH) analysis of wdr68 and dyrk1b mutants and morphants to determine whether there are defects in Hh signaling. We will also treat embryos with small molecule activators and inhibitors of the Hh signaling pathway for potential restoration of jaw development. We expect to see altered levels of at least some aspects of Hh signaling in mutants and morphants. We also expect Hh antagonists will restore cartilage formation in wdr68 mutants. Second, we will test whether ectopic endothelin-1 (edn1) expression can rescue the lower jaw defects in wdr68 mutant or morphant embryos. We hypothesize that ectopic edn1 expression in wdr68 mutants will restore lower jaw formation. The experimental approach will employ the parallel approaches of edn1 expression construct injection as well as the use of a GAL4-UAS system to overexpress edn1 in wdr68 morphants. We expect that ectopic edn1 expression will restore lower jaw formation in wdr68 mutants/morphants. Third, we will determine the temporal requirement for wdr68 activity in craniofacial development. We hypothesize that wdr68 is required during late-somitogenesis stages for normal craniofacial development. The experimental approach will use a heat shock inducible GFP- Wdr68 Tg(hsp70l:GFP-Wdr68) transgenic line. Because several gene expression defects are readily detected by 24hpf, we expect slightly earlier (by 18hpf) heat shock induction of GFP-Wdr68 to rescue transgenic wdr68- MO animals while late heat shock (20hpf or later) to fail to rescue craniofacial development.